Properties of Formaldehyde Dismutation Catalyzing Enzymeof

Agric. Biol. Chem., 48 (8), 2017-2023, 1984 2017 Properties of Formaldehyde Dismutation Catalyzing Enzymeof Pseudomonas putida F61 Nobuo Kato, Hisataka Kobayashi, Masayuki Shimao and Chikahiro Sakazawa Department of Environmental Chemistry and Technology, Tottori University, Tottori 680, Japan Received January 9, 1984 Twoforms of formaldehyde dismutase distinguishable on disc-gel electrophoresis were isolated from the cell-free extract of Pseudomonasputida ¥61. The mobilities on SDS-gel electrophoresis and the NH2-terminal amino acids (arginine) of the two enzyme species were identical. The COOH- terminal amino acid sequence was found to be -Ser-Gly-Lys. The enzyme was inhibited by carbonyl, reducing and sulfhydryl reagents. The enzyme catalyzed the cross-dismutation reaction between formaldehyde and an aldehyde, such as propionaldehyde, acrolein, butyraldehyde, isobutyraldehyde and crotonaldehyde. The enzyme also catalyzed a coupled oxidoreduction between an alcohol and an aldehyde (RCH2OH+ R CHO^RCHO+R CH2OH) without addition of an electron acceptor. Aliphatic alcohols and aldehydes of C2 to C4 were utilized in this reaction. In the preceding study,1} we found an en- (E; formaldehyde dismutase, X; unknown zyme, which catalyzed dismutation of form- prosthetic group, and XH2; its reduced form.) aldehyde to form equimolar amounts of meth- Furthermore, we have found that the enzyme anol and formic acid, in Pseudomonas putida catalyzes a unique alcohol: aldehyde oxido- F61. The enzyme, given the trivial name of reduction reaction: formaldehyde dismutase, was purified and RCH2OH+R CHO >RCHO+R CH2OH partially characterized. On the other hand, mammalian alcohol dehydrogenase (EC In this work, we describe more detailed 1.1.1.1) was reported to catalyze formalde- properties of this enzyme and its substrate hyde dismutation on the addition of a cata- specificities in the cross-dismutation and al- lytic amount of NAD+or one of its deriva- cohol: aldehyde oxidoreduction reactions. tives.2) The enzyme from P. putida F61, which is distinct from the mammalian one, catalyz- MATERIALS AND METHODS ed the dismutation without addition of an electron acceptor, such as a pyridine nucleo- Organism and cultivation. P. putida F61 was grown on a tide coenzyme or artificial dye.X) Another uni- nutrient medium as reported previously.1* The harvested que property of the formaldehyde dismutase cells were washed twice with lOmMpotassium phosphate buffer, pH 7.0, and the cell paste was stored at -15°C was the catalytic activity of cross-dismutation until use. between two different aldehydes: RCHO"+H2O+Eà"X >RCOOH+Eà"XH2 Enzymeassay. Formaldehyde dismutase was assayed by determination of formic acid formed by means of pHstat R CHO+Eà"XH2 >R CH2OH+Eà"X titration with NaOHat pH 7.O.1* Protein determination. Protein was estimated by the Bio- RCHO+H2O+R CHO > Rad Protein Assay (Bio-Rad Laboratories) with bovine RCOOH+R CH2OH serum albumin as the standard. The protein concentration of the purified enzyme was determined with an 2018 N. Kato et al. 4280nmvalue of 8.2 which was obtained from the absor- raise the yield of the purified enzyme, the purification bance and dry weight determinations. procedures were modified as follows. Amino acid determination. For determination of the (0 Acetone fractionation. The protamine-treated en- amino acid composition, the purified enzyme was hy- (v/v)zymepreparationwith cold acetone(20.6 g(-20°C),as protein)and wasthe broughtresulting to pre-30% drolyzed in 6n HC1 containing 2% thioglycolic acid at cipitate was removedby centrifugation. Cold acetone was 105°C for 20~70'hr in an evacuated sealed tube. Before added to the supernatant solution to 70%. The precipitate hydrolysis, norleucine was added to the enzymesolution collected by centrifugation was dissolved in 100ml of to the concentration of 0.5 /miol/mg protein as an internal 10mMpotassium phosphate buffer (pH 7.0) and dialyzed standard. The hydrolyzate was analyzed with an amino for 18hr against 10 liters of the same buffer. acid autoanalyzer (Kyowa Seimitsu Co., type K-101). (ii) DEAE-Sephacel column chromatography. The dia- Cysteine and cystine were determined as cysteic acid after lyzed solution (5.7g as protein) was applied to a DEAE- performate oxidation of the sample according to Hirs.3) Sephacel column (2.5 by 45 cm). The column was washed Tryptophan was determined spectrophotometrically.4) once with 2.5 liters of the equilibration buffer, lOmM The NH2-terminal amino acid was determined by the potassium phosphate (pH 7.0), and then with 2 liters of the dansyl method according to Ikenaka.5) To identify the buffer containing 100mMNaCl. The enzyme was eluted dansyl amino acids produced during acid hydrolysis, thin with a linear gradient of increasing NaCl concentration layer chromatography on Polyamide Layer Sheets between 100 and 300mM (total volume, 2.5 liters). The (Seikagaku Kogyo) was performed. A spotted sheet was active fractions collected and pooled were dialyzed for sequentially developed with four kinds of solvents accord- 24hr against 3 changes of a 10 liter volume of 10mM ing to Woods and Wang.6) The COOH-terminal amino phosphate buffer, pH 7.0, and concentrated to a one- acid was determined with carboxypeptidases (CPase) A tenth volume by ultra filtration with a Minimodule and B (Sigma Chemical Co., type, I-DFP), the molar (Asahikasei Co., Ltd.). ratio of which to the formaldehyde dismutase was 1/ Through the purification procedures, the enzyme was 25.7) The released amino acids were determined with the purified about 22-fold from the cell-free extract of P. autoanalyzer mentioned above. putida F61 (Table I). The enzyme yield after DEAE- Sephacel chromatography was two times higher than that Electrophoresis. Polyacrylamide gel electrophoresis with the previous methods.1* was carried out in 5%polyacrylamide gels in Tris-glycine (Hi) Phenyl-Sepharose CL-4B column chromatography. buffer (pH 8.3) according to Davis.8) The zone of the To the concentrated solution (50ml, 256mg as protein) formaldehyde dismutase activity was visualized by in- was added solid NaCl to a concentration of 4m, and the cubating the gels for 60min at 30°C in 5ml of a reaction mixture was applied to a Phenyl-Sepharose column (2.5 by mixture containing 50mMpotassium phosphate buffer 20 cm) equilibrated with lO mMpotassium phosphate (pH (pH 7.5), 20mM formaldehyde, 0.2mM NAD+, 0.02mg of 7.0) containing 4mNaCl. Elution was carried out with a phenazine methosulfate per ml, 1 mg 2,3,5-triphenyltetra- gradient of decreasing NaCl concentration and increasing zolium chloride per ml, and 2 units of formate dehy- ethylene glycol concentration (the final concentrations drogenase per ml. The reaction was stopped by adding were 0 and 50%, respectively; total volume, 500ml). The 0.5ml of acetic acid and then the gels were removed and elution pattern of the enzyme is described below. The stored in 7% (v/v) acetic acid. The electrophoresis on polyacrylamide gels containing sodium dodecylsulfate withactivea fractionsMinimodule,collectedand dialyzedand pooledforwere24hrconcentratedagainst 3 (SDS) was carried out according to Shapiro et al.9) changes of a 10 liter volume of 10mMpotassium phosphate buffer (pH 7.0). Gas chromatography. Alcohols, aldehydes and acids were determined by gas chromatography (Shimadzu Preparative polyaerylamide gel electrophoresis. A 0.5-ml GC-8A) with a flame ionization detector. A glass col- aliquot of the enzyme solution (5 mgprotein) obtained at umn (0.26x200cm) was filled with 80/100 mesh Gasu- the Phenyl-Sepharose step was applied to a slab gel (16 by kuropack 54 (Gasukuro Kogyo Inc.). The temperatures 14cm, 2mmthick) which was in contact with a cooling of the column and inlet were maintained at 180 and plate, and electrophoresis was carried out at 15mAac- 200°C, respectively. The flow rate of nitrogen carrier cording to Bringer et al.10) After the electrophoresis, gas was 60ml/min. The reaction mixture was directly in- the gel was blotted with a filter paper (Toyo Roshi No. jected into the column. 51A), which was immediately stained with Coomassie brilliant blue. The gel zone corresponding to the stained Enzyme purification. As described previously,1* the en- protein band on the paper was cut out and crushed with zyme waspurified from the cell-free extract of P. putida a teflon homogenizer. From the disintegrated gel, the en- F61 by protamine treatment, ammoniumsulfate fraction- zyme was eluted with 10mMphosphate buffer (pH 7.0) ation, and chromatographies on Phenyl-Sepharose CL- with stirring overnight. The polyacrylamide was remov- 4B, DEAE-Sephacel and hydroxyapatite. In order to ed by centrifugation, and the supernatant wasdialyzed Formaldehyde Dismutation Catalyzing Enzyme 2019 Table I. Purification of the Formaldehyde Dismutase from Pseudomonas putida F61 Total Total Specific Step protein activity activity Purification f (mg) (U) (U/mg) ( /o) Cell-free extract 21 ,400 90,000 4.21 1.0 100 Protamine sulfate 20,600 87,200 4.23 1.0 96.9 Acetone fractionation 5,700 52,600 9.23 2.2 58.4 DEAE-Sephacel 256 24, 100 91. 1 21.6 26.8 against lOmMphosphate buffer (pH 7.0), and then concentrated with an Immersible CX-10 ultra filter (Millipore Corp.) to 0.5ml. RESULTS Protein nature of the purified enzyme As shownin Fig. 1, two active fractions of formaldehyde dismutase appeared on Phenyl- Sepharose column chromatography as de- ; v...^ 100 150 scribed under Materials and Methods. The 20 * å : 10ml/fraction first fraction (A; fractions 104~130) was Fig. 1. Phenyl-Sepharose CL-4B Column Chromatog- identical with the enzyme which was purified raphy of the Formaldehyde Dismutase. previously. The specific activity of the latter The details are given in Materials and Methods. ----, fraction (B; fractions 183-234), 92/miol/ NaCl; - --, ethylene glycol. minà"mg,was about half of that of the A-fraction. On polyacrylamide gel electrophoresis, the A-fraction preparation gave two distinct Molecular weight and subunit of the B-fraction protein bands (A-l and A-2) whose migra- enzyme tion rates were 0.38 and 0.44, respectively.

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